体细胞直接重编程为神经元和神经干细胞

体细胞直接重编程是由已分化细胞类型不经过诱导型多能干细胞(Induced pluripotent stem cells,i PSCs)中间阶段,直接转换为另一种细胞类型的重编程过程。体细胞直接重编程避免了i PSC技术存在的重编程效率低下、引入致癌基因等多种缺陷,并为细胞替换治疗和个性化医药研发设想贡献了新的实现途径。现代医学对于诸如神经退行性疾病、神经遗传疾病和外伤导致的神经细胞受损等一些神经系统疾病一直没有有效的治疗手段。而体细胞直接重编程为治疗这些疾病提供了另一种治疗途径,因此体细胞直接重编程为神经细胞相关领域迅速成为研究热点。回顾了体细胞重编程为诱导型神经元(Induced neurons,i Ns)和诱导型神经干细胞(Induced neural stem cells,i NSCs)的最新研究进展,并探讨i Ns和i NSCs在临床应用上的各自优势、局限性及应用前景。     

胚胎干细胞体外分化为多巴胺能神经元

近年来,胚胎干细胞在体外分化为多巴胺能神经元方面取得了重大突破,这对神经发生的基础性研究和神经细胞移植具有重要意义。现对胚胎干细胞体外定向诱导分化为多巴胺能神经元的方法、相关细胞因子及检测鉴定等方面进行了分析和比较,并探讨了当前存在的问题和今后发展的方向。     

间充质干细胞体外分化为多巴胺能神经元的研究进展

骨髓间充质干细胞（MSCs）有来源广泛、易于分离培养、不易引起免疫排斥等特点,使其成为细胞治疗和基因治疗的种子细胞,具有广泛的科研和临床应用价值。骨髓MSCs具有多向分化潜能,在特定条件下能诱导分化成神经元甚至是更为特异的多巴胺能神经元,为帕金森病进行细胞移植疗法提供了理想的细胞来源。本文就近年来体外诱导MSCs向多巴胺能神经元定向分化所涉及到的常用诱导因素和诱导方法及途径予以综述。     

神经干细胞向神经元分化调控的研究进展

神经干细胞是一类具有分裂潜能和自更新能力的母细胞,它可以通过对称分裂和不对称分裂方式产生神经组织的各类细胞,包括神经元、星形胶质细胞和少突胶质细胞。中枢神经系统受到损伤后,神经元和胶质细胞的损伤导致了临床症状,内源性神经干细胞的修复作用不大,原因是干细胞的数量有限,微环境的不允许。移植的神经干细胞进入体内后,由于受到多种因素的影响,常保持未分化状态或大部分分化为胶质细胞。神经干细胞向神经元分化的调控机制及其影响因素直接决定神经干细胞源性神经元的比例和神经元之间功能性突触的数量。现就其研究进展做一综述。     

小鼠胚胎干细胞在单层粘附培养中向神经细胞的分化

目的 :探讨小鼠胚胎干 (ES)细胞在无血清培养基中以单层粘附培养方式向神经分化的方法。方法 :比较ES细胞在不同培养基中的生长情况 ,分析ES细胞在不同时间分化形成神经细胞的比例。结果 :( 1 )DMEM F1 2和Neurobasal B2 7的 1∶1混合培养基最适合ES的生长。 ( 2 )单层粘附的ES细胞表达神经细胞粘附分子 (NCAM)的比例随时间增长而增加 ,而nestin的表达先增加后下降。 ( 3)ES细胞可在两周分化为神经胶质及神经元 ,形成神经网络。结论 :小鼠ES细胞可在单层粘附培养中获得向神经的高效分化。     

Repression of SIRT1 Promotes the Differentiation of Mouse Induced Pluripotent Stem Cells into Neural Stem Cells

The use of transplanting functional neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) has increased for the treatment of brain diseases. As such, it is important to understand the molecular mechanisms that promote NSCs differentiation of iPSCs for future NSC-based therapies. Sirtuin 1 (SIRT1), a NAD⁺-dependent protein deacetylase, has attracted significant attention over the past decade due to its prominent role in processes including organ development, longevity, and cancer. However, it remains unclear whether SIRT1 plays a role in the differentiation of mouse iPSCs toward NSCs. In this study, we produced NSCs from mouse iPSCs using serum-free medium supplemented with retinoic acid. We then assessed changes in the expression of SIRT1 and microRNA-34a, which regulates SIRT1 expression. Moreover, we used a SIRT1 inhibitor to investigate the role of SIRT1 in NSCs differentiation of iPSCs. Data revealed that the expression of SIRT1 decreased, whereas miRNAs-34a increased, during this process. In addition, the inhibition of SIRT1 enhanced the generation of NSCs and mature neurocytes. This suggests that SIRT1 negatively regulated the differentiation of mouse iPSCs into NSCs, and that this process may be regulated by miRNA-34a.     

In Vitro Differentiation of Mouse Embryonic Stem Cells into Neurons of the Dorsal Forebrain

Pluripotent embryonic stem cells (ESCs) are able to differentiate into all cell types in the organism including cortical neurons. To follow the dynamic generation of progenitors of the dorsal forebrain in vitro, we generated ESCs from D6-GFP mice in which GFP marks neocortical progenitors and neurons after embryonic day (E) 10.5. We used several cell culture protocols for differentiation of ESCs into progenitors and neurons of the dorsal forebrain. In cell culture, GFP-positive cells were induced under differentiation conditions in quickly formed embryoid bodies (qEBs) after 10–12 day incubation. Activation of Wnt signaling during ESC differentiation further stimulated generation of D6-GFP-positive cortical cells. In contrast, differentiation protocols using normal embryoid bodies (nEBs) yielded only a few D6-GFP-positive cells. Gene expression analysis revealed that multiple components of the canonical Wnt signaling pathway were expressed during the development of embryoid bodies. As shown by immunohistochemistry and quantitative qRT-PCR, D6-GFP-positive cells from qEBs expressed genes that are characteristic for the dorsal forebrain such as Pax6, Dach1, Tbr1, Tbr2, or Sox5. qEBs culture allowed the formation of a D6-GFP positive pseudo-polarized neuroepithelium with the characteristic presence of N-cadherin at the apical pole resembling the structure of the developing neocortex.     

人胚胎干细胞定向分化为神经前体细胞

采用单层贴壁分化的方法在无血清条件下诱导同源饲养层培养的人胚胎干细胞定向分化,得到了高比例的神经前体细胞(97.5±0.83)%(P<0.05)。这些神经前体细胞具有分化为神经元、星形胶质细胞和少突胶质细胞的能力。在长期的传代培养中发现,随着培养时间的延长,nestin阳性的神经前体细胞比例下降,同时发育能力也发生了变化。在传代培养的早期,神经前体细胞发育为神经元的比例很高,几乎没有胶质细胞分化出来。随着培养时间的延长,胶质细胞的比例逐渐上升。这与体内神经系统的发育过程非常相似。进一步研究发现具有bHLH(basic helix-loop-helix)结构域的转录因子neurogenein2(Ngn2)和Olig2可能在这一变化中起重要作用。因此,人胚胎干细胞来源的神经前体细胞能够模拟体内神经发育的模式,为在体外研究人的神经发育和再生医学奠定了基础。     

NEUROD1 Intrinsically Initiates Differentiation of Induced Pluripotent Stem Cells into Neural Progenitor Cells

Cell type specification is a delicate biological event in which every step is under tight regulation. From a molecular point of view, cell fate commitment begins with chromatin alteration, which kickstarts lineage-determining factors to initiate a series of genes required for cell specification. Several important neuronal differentiation factors have been identified from ectopic over-expression studies. However, there is scarce information on which DNA regions are modified during induced pluripotent stem cell (iPSC) to neuronal progenitor cell (NPC) differentiation, the cis regulatory factors that attach to these accessible regions, or the genes that are initially expressed. In this study, we identified the DNA accessible regions of iPSCs and NPCs via the Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq). We identified which chromatin regions were modified after neuronal differentiation and found that the enhancer regions had more active histone modification changes than the promoters. Through motif enrichment analysis, we found that NEUROD1 controls iPSC differentiation to NPC by binding to the accessible regions of enhancers in cooperation with other factors such as the Hox proteins. Finally, by using Hi-C data, we categorized the genes that directly interacted with the enhancers under the control of NEUROD1 during iPSC to NPC differentiation.     

神经干细胞脑内移植后的存活、迁移和分化

从胚胎或成体大鼠脑组织、人胚脑组织均能分离到神经干细胞 ,将它们进行体外原代培养扩增或永生化后植入脑内 ,均能观察到其在脑内的迁移和分化现象。其分化能力主要取决于移植部位的脑内微环境 ,但这种影响作用是相对的。同时 ,体外培养环境如培养时间和细胞融合程度、维甲酸类诱导分化剂处理、NGF转导处理再移植或与嗜铬细胞 (分泌NGF)共移植等 ,也能决定神经干细胞脑内移植后向神经元方向分化的能力。神经干细胞移植为中枢神经系统功能重建和神经再生带来新的希望。     

丙戊酸影响神经干细胞体外分化的量效和时效关系

目的：研究丙戊酸（VPA）浓度和干预时间对神经干细胞（NSCs）体外分化的影响。方法：以不同浓度的VPA（0.1、0.3、0.5、0.75和1.0mmol/L）处理原代培养的神经干细胞,以NB培养基组做对照,分别于神经干细胞分化后3天、7天、10天和14天用免疫荧光双标鉴定并计数微管蛋白-Ⅲ（β-tubllin III）和胶质原纤维酸性蛋白（GFAP）阳性细胞的比例,并作统计学分析。结果：同一时相点组间比较,3天时各组中神经元分化比例无显著差异;7天时不同浓度VPA组与对照组分化神经元比例开始呈现差异;10天时这种差异继续增大,0.75 mmol/L VPA组中神经元比例为82.15±0.93%;14天时保持这种差异;但各时相点1.0 mmol/L VPA组与0.75 mmol/L VPA组神经元分化的比例无显著差异。不同时相点组内比较发现,3-10天内随着时间的延长,各组神经元分化的比例显著增加,但14天时神经元分化的比例较10天无显著变化。结论：0.75mmol/L VPA在10天时促神经干细胞向神经元分化的作用最佳。     

丙戊酸影响神经干细胞体外分化的量效和时效关系

目的:研究丙戊酸(VPA)浓度和干预时间对神经干细胞(NSCs)体外分化的影响。方法:以不同浓度的VPA(0.1、0.3、0.5、0.75和1.0mmol/L)处理原代培养的神经干细胞,以NB培养基组做对照,分别于神经干细胞分化后3天、7天、10天和14天用免疫荧光双标鉴定并计数微管蛋白-Ⅲ(β-tubllin III)和胶质原纤维酸性蛋白(GFAP)阳性细胞的比例,并作统计学分析。结果:同一时相点组间比较,3天时各组中神经元分化比例无显著差异;7天时不同浓度VPA组与对照组分化神经元比例开始呈现差异;10天时这种差异继续增大,0.75 mmol/L VPA组中神经元比例为82.15±0.93%;14天时保持这种差异;但各时相点1.0 mmol/L VPA组与0.75 mmol/L VPA组神经元分化的比例无显著差异。不同时相点组内比较发现,3-10天内随着时间的延长,各组神经元分化的比例显著增加,但14天时神经元分化的比例较10天无显著变化。结论:0.75mmol/L VPA在10天时促神经干细胞向神经元分化的作用最佳。     

胚胎干细胞向肝细胞诱导分化

胚胎干细胞具有分化成三胚层细胞的潜能。它已被视为治疗多种疾痛的一种新兴策略。在现阶段,通过不同的诱导途径可将胚胎干细胞诱导成为肝细胞：体外诱导、体内诱导以及体外和体内相结合诱导分化。然而从体内实验结果来看,其嵌合率及分化率不高,这是一个亟需解决的问题,否则就无法成功地将其应用于临床治疗。     

成体干细胞向肝细胞分化

成体干细胞跨越胚层限制分化为其他胚层来源的细胞,对揭示不同胚层细胞间相互分化的生物学意义和机制具有重要学术价值,并可以为临床细胞移植治疗开辟新的途径,从而成为当前研究的热点之一。综述了近年来肝源性卵圆细胞、成肝细胞、骨髓源干细胞和其他成体干细胞跨越分化为肝细胞的研究现状与进展,以及卵圆细胞、成肝细胞等的分离鉴定,表面标志、生物学特征和跨越分化机制,并对成体干细胞在肝脏疾病细胞治疗上的应用前景作了展望。     

DNA甲基化修饰与干细胞分化

干细胞自我更新及分化潜能一方面是内源性转录因子相互协调控制的结果,另一方面表观遗传修饰也起着重要的作用。该文综述了DNA甲基化修饰的机理、哺乳动物DNA甲基化的特点以及干细胞分化的DNA甲基化修饰。     

The Effects of Co-Culture of Embryonic Stem Cells with Neural Stem Cells on Differentiation

Researching the technology for in vitro differentiation of embryonic stem cells (ESCs) into neural lineages is very important in developmental biology, regenerative medicine, and cell therapy. Thus, studies on in vitro differentiation of ESCs into neural lineages by co-culture are expected to improve our understanding of this process. A co-culture system has long been used to study interactions between cell populations, improve culture efficiency, and establish synthetic interactions between populations. In this study, we investigated the effect of a co-culture of ESCs with neural stem cells (NSCs) in two-dimensional (2D) or three-dimensional (3D) culture conditions. Furthermore, we examined the effect of an NSC-derived conditioned medium (CM) on ESC differentiation. OG2-ESCs lost the specific morphology of colonies and Oct4-GFP when co-cultured with NSC. Additionally, real-time PCR analysis showed that ESCs co-cultured with NSCs expressed higher levels of ectoderm markers Pax6 and Sox1 under both co-culture conditions. However, the differentiation efficiency of CM was lower than that of the non-conditioned medium. Collectively, our results show that co-culture with NSCs promotes the differentiation of ESCs into the ectoderm.     

Histone Deacetylase Inhibitor Valproic Acid Promotes the Differentiation of Human Induced Pluripotent Stem Cells into Hepatocyte-Like Cells

In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells.     

Behavior and Differentiation of the Neural Stem Cells in vivo

We studied the behavior and differentiation of human and rat neural stem cells after transplantation in the adult rat brain without immunosuppression. The rat stem cells were isolated from the presumptive neocortex of 15-day-old embryos. The human cells were isolated from the ventricular brain zone of 9-week-old embryos and cultivated for two weeks before transplantation. The results of histomorphological studies suggest that the microenvironment factors did not suppress the growth or development of transplanted stem cells. Both rat and human embryonic multipotent neural cells showed similar behavior and differentiation into neurons and glial cells. After transplantation, they continued to mitotically divide and migrated from the graft area to the surrounding tissue of a recipient brain. The presumptive glial cells migrated preferentially along the capillaries and fibrous structures of the recipient brain. Similar behavior of the rat and human neural stem cells in the microenvironment of the recipient adult rat brain and the absence of immune reaction suggest that the transplantation into the rat brain may serve as a model for studying the developmental biology of the human stem cells.